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. 2024 Jan 12;5(4):100817. doi: 10.1016/j.xplc.2024.100817

Suppression of host plant defense by bacterial small RNAs packaged in outer membrane vesicles

Yan Wu 1, Sai Wang 1, Peihong Wang 1, Wenhan Nie 1, Iftikhar Ahmad 1,2, Panagiotis F Sarris 3, Gongyou Chen 1,∗, Bo Zhu 1,4,∗∗
PMCID: PMC11009154  PMID: 38217288

Abstract

Noncoding small RNAs (sRNAs) packaged in bacterial outer membrane vesicles (OMVs) function as novel mediators of interspecies communication. While the role of bacterial sRNAs in enhancing virulence is well established, the role of sRNAs in the interaction between OMVs from phytopathogenic bacteria and their host plants remains unclear. In this study, we employ RNA sequencing to characterize differentially packaged sRNAs in OMVs of the phytopathogen Xanthomonas oryzae pv. oryzicola (Xoc). Our candidate sRNA (Xosr001) was abundant in OMVs and involved in the regulation of OsJMT1 to impair host stomatal immunity. Xoc loads Xosr001 into OMVs, which are specifically ttransferred into the mechanical tissues of rice leaves. Xosr001 suppresses OsJMT1 transcript accumulation in vivo, leading to a reduction in MeJA accumulation in rice leaves. Furthermore, the application of synthesized Xosr001 sRNA to the leaves of OsJMT1-HA-OE transgenic line results in the suppression of OsJMT1 expression by Xosr001. Notably, the OsJMT1-HA-OE transgenic line exhibited attenuated stomatal immunity and disease susceptibility upon infection with ΔXosr001 compared to Xoc. These results suggest that Xosr001 packaged in Xoc OMVs functions to suppress stomatal immunity in rice.

Key words: small RNAs, outer membrane vesicles, Xanthomonas oryzae, stomatal immunity, cross-species communication

This study reports that the sRNA Xosr001, delivered into epidermal and mechanical tissues of rice leaves through OMVs secreted by Xoc BLS256, suppresses OsJMT1 transcript accumulation and MeJA accumulation to repress stomatal immunity. These findings advance our understanding of the mechanisms by which bacterial nucleic acids are transported between species to modulate host immune responses.

Introduction

Bacterial outer membrane vesicles (OMVs) play an essential role in interspecies and interkingdom interactions, and are continuously being formed and released from the outermost membrane throughout the bacterial life cycle (Cho and Blaser, 2012; Schwechheimer and Kuehn, 2015; Liu et al., 2022). OMVs are nanospherical proteoliposomes with a diameter ranging from 20 to 400 nm. They contain proteins, lipids, nucleic acids, and other molecules that are delivered to host cells to suppress host immunity (Gill et al., 2019; Sedaghat et al., 2019). Bacteria can internalize small RNAs (sRNAs) within OMVs, which then fuse to the host plasma membrane and deliver the sRNA cargo into the cytoplasm to help establish interspecies communication (Koeppen et al., 2016; Choi et al., 2017). The ability of sRNAs to regulate host transcript stability suggests they could play an important role in bacterial-host interactions. Bacterial sRNAs exert multiple biological functions, including environmental adaptation, quorum sensing, bacterial communication, and virulence. They do this primarily by regulating target gene transcript abundance through complementary base pairing (Dötsch et al., 2012; Shao and Bassler, 2012; Wurtzel et al., 2012).

Several studies have confirmed that host immune responses triggered by sRNAs packaged in OMVs favor both in vivo bacterial growth and host infection (Koeppen et al., 2016; Lee, 2019; Moriano-Gutierrez et al., 2020; Liu et al., 2021). For example, SsrA is an sRNA that is specifically delivered by OMVs into epithelial cells of the Hawaiian bobtail squid (Euprymna scolopes) where it regulates the retinoic acid-inducible gene-I-like receptor signaling pathway in Vibrio fischeri (Moriano-Gutierrez et al., 2020). Similarly, sRNA52320 is delivered by Pseudomonas aeruginosa OMVs into human epithelial airway cells where it leads to reduced LPS-induced IL-8 secretion (Koeppen et al., 2016).

Most studies on OMVs have focused on the bacterial pathogens of animals, whereas the role of OMVs in interactions between phytopathogenic bacteria and host plants remains largely unexplored. OMVs produced by phytopathogenic bacteria can either promote bacterial replication within the plant host or activate host immune responses. Xylella fastidiosa OMVs contribute to pathogen virulence by promoting bacterial attachment to the walls of xylem vessels (Ionescu et al., 2014). Conversely, Pseudomonas syringae pv. tomato DC3000 OMVs elicit plant immune responses that confer immunity against both fungal and bacterial pathogens (Janda et al., 2021; McMillan et al., 2021). A recent study demonstrated that OMVs produced by Xanthomonas campestris pv. campestris (Xcc) can trigger an immune response in Arabidopsis by inserting lipids into the plasma membrane (Tran et al., 2022). However, the role of sRNAs delivered by OMVs in plant-microbe interactions is poorly understood.

Xanthomonas oryzae pv. oryzicola (Xoc) causes bacterial leaf streak in rice by infecting leaves through stomata and wounds (Niño-Liu et al., 2006). Stomatal immunity is activated during the early stages of bacterial infection (Melotto et al., 2017). Phytopathogenic bacteria secrete effectors or phytotoxins to suppress stomatal immunity. For example, the Xoc type III effector XopC2 activates jasmonate (JA) signaling to open stomata and facilitate bacterial entry into rice leaves (Wang et al., 2021).

In the present study, we explore the mechanism by which sRNAs packaged in OMVs from the Gram-negative plant pathogen Xoc BLS256 are loaded into host plant cells and the subsequent impact of these sRNAs on plant immune responses. OMVs released from Xoc BLS256 were extracted using ultracentrifugation and purified using density gradient centrifugation. Four candidate sRNAs were identified using RNA sequencing (RNA-seq) and northern blotting. We further characterized the most abundant sRNA from Xoc BLS256, Xanthomonas OMV sRNA 001 (Xosr001), and revealed that it is transferred from OMVs into epidermal and mechanical tissues, as well as phloem cells of rice leaves. Xosr001 suppressed OsJMT1 transcript accumulation and reduced methyl jasmonate (MeJA) accumulation in rice leaves. Synthetic Xosr001 applied exogenously to rice leaves also suppressed the expression of an OsJMT1 transgene in the OsJMT1-HA-OE transgenic line. OsJMT1 was overexpressed and knocked out in the Nipponbare rice cultivar to study the impact of Xosr001 on OsJMT1 transcription. Stomatal conductance and spray inoculation assays demonstrated that infection by Xoc BLS256-producing OMVs lacking Xosr001 were significantly restricted during the very early stage of infection. Conversely, Xoc BLS256-producing OMVs containing Xosr001 were able to inhibit stomatal immunity and promote infection of rice leaves.

Results

OMVs contain differentially packaged sRNAs

Xoc BLS256 OMVs were isolated using ultracentrifugation and purified using OptiPrep gradient density centrifugation. Transmission electron microscopy (TEM) analysis revealed the presence of distinct spheroid particles with an envelope structure and electron-dense luminal contents characteristic of OMVs (Figure 1A). A nanoparticle tracking analysis revealed that the OMVs released by Xoc BLS256 ranged in size from 20 to 400 nm and had an average diameter of 120 nm (Figure 1B). These results confirmed the successful extraction of Xoc BLS256 OMVs. An RNA-seq analysis was performed on purified Xoc BLS256 OMVs to determine if sRNAs are packaged in OMVs from this pathogen. The results revealed that most RNA species were ribosomal RNAs (58.03%), while tRNAs (21.79%) and small ncRNAs (2.16%) were also present (Supplemental Figure 1). The RNA-seq analysis also identified 3615 unique sRNA sequences present in OMVs, with the eight most abundant candidate sRNAs being designated as Xosr001-Xosr008. The cDNA sequences of the intergenic sRNAs Xosr001, Xosr002, Xosr003, and Xosr006 were cloned for further validation (Figure 1C and Supplemental Table 3). A northern blot analysis was used to confirm the transcript lengths of the four sRNA candidates tested previously (Figure 1D).

Figure 1.

Figure 1

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Xoc BLS256 releases OMVs enriched in sRNAs

(A) Transmission electron microscopy image of OMVs purified from Xoc BLS256 following density step-gradient centrifugation. Scale bar corresponds to 200 nm.

(B) Size distribution of OMVs detected by nanoparticle tracking analysis.

(C) PCR products of cDNA obtained in RT-PCR and fractionated by 3% agarose gel electrophoresis. Total RNA was isolated from purified Xoc BLS256 OMVs using QIAzol reagent, and a Magic First cDNA Synthesis Kit (Magic-Bio) was used to generate cDNA.

(D) Northern blots verified the presence of Xoc BLS256 OMV sRNAs. Transcript sizes are approximate and compared to digoxygenin-labeled marker (M).

Xosr001 is transferred from OMVs to host plant cells

(Supplemental Figure 1) The mapped reads fully covered the sRNA sequences, indicating the sRNAs were not degraded. Xosr001 was the most abundant sRNA identified and is a homolog of sRX061(Supplemental Table 3), which was previously shown to play an important role in the virulence of Xcc by Tang et al. (2020). We predicted the secondary structure of Xosr001 using software available at https://sfold.wadsworth.org (Supplemental Figure 2). OMVs were treated with RNase A, a membrane-impermeable enzyme that only degrades sRNAs adhered to the exterior of OMVs, to determine if Xosr001 was packaged inside OMVs (Supplemental Figure 3A). A northern blot was performed on RNA isolated from OMVs treated with RNase A, Triton X-100, or untreated OMVs. More Xosr001 sRNAs were isolated from OMVs treated with RNase A than untreated OMVs, and many Xosr001 sRNAs were isolated from OMVs treated with Triton X-100 (Supplemental Figure 3B). These results suggest that Xosr001 sRNA was packaged within OMVs.

This study revealed that Xoc BLS256 is prolific producer of OMVs. TEM revealed the presence of OMV-like nanoparticles in rice leaves after spray inoculation with RNase A-treated OMVs at 16 and 24 h (Figure 2A), indicating OMVs can enter rice leaves through stomata. To confirm that Xosr001 enters host plant tissues via OMVs, RNA fluorescence in situ hybridization (RNA-FISH) was employed. As demonstrated in Figure 2, Xosr001 transcripts were detected in leaves inoculated with RNase A-treated OMVs from wild-type (WT) Xoc BLS256 after 24 h (Figure 2B), whereas no signal was detected when Triton X-100-treated OMVs were used to inoculate leaves (Figure 2B). Notably, Xosr001 sRNAs localized in the epidermal and mechanical tissues of rice leaves (Figure 2). We concluded that the epidermal and mechanical tissues of rice leaves may be particularly susceptible to bacterial cargo, such as Xosr001 from Xoc BLS256, delivered by symbiont-derived OMVs.

Figure 2.

Figure 2

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Xosr001 localizes within epidermal and mechanical tissues of rice leaves

Transmission electron microscopy images of leaves from 6-week-old Nipponbare seedlings after spray inoculation with purified OMVs from Xoc BLS256 for 16 h (A) and 24 h (B). Localization of the Xosr001 transcript viewed by confocal microscopy 24 h after spray inoculation with OMVs treated with 10 pg/μl RNase A and 0.5% Triton X-100. Left: merged images with orthogonal views; other panels: images of individual labels.

OMV-packaged Xosr001 triggers host plant responses

An increase in plant defense gene expression is an important and early response that triggers defense responses in infected tissue and serves as a signal for intercellular transmission in response to infection (Bell et al., 1986). To examine the influence of Xosr001 on plant defense responses, we created a clean-deletion of Xosr001 in the Xoc BLS256 background (ΔXosr001) and performed a comparative RNA-seq analysis on rice leaves inoculated with WT and ΔXosr001 at 24 h post-inoculation (hpi). Compared to leaves inoculated with the WT strain, host genes associated with the phytohormone synthesis pathway and stress-responsive genes with known immune functions were significantly upregulated in leaves inoculated with the ΔXosr001 strain (Figure 3A).

Figure 3.

Figure 3

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Xosr001 impacts the expression of OsJMT1

Host responses to colonization differ by WT or ΔXosr001.

(A) Heatmap depicting fold-change differences in significantly differentially expressed genes in leaves of Nipponbare rice plants colonized by the WT strain and the ΔXosr001 mutant. Genes that are upregulated in ΔXors001-colonized leaves compared to WT-colonized leaves are indicated in bold. The replicate number for each condition is indicated beneath the heatmap.

(B) Northern blot analysis of Xosr001 and OsJMT1 transcript levels determined by inoculating 6-week-old rice leaves with Xoc BLS256 for 0, 10, 18, 24, 36, or 48 h post-inoculation (hpi). 5S rRNA and OsActin were used as loading controls. The values above each band represent band intensity and were calculated using ImageJ software. Band intensity in the first lane is normalized to 100.

(C) Expression levels of OsJMT1 after inoculation with WT, ΔXosr001, and ΔXosr001-pXosr for 0, 10, 18, 24, 36, or 48 hpi. Gene expression was detected using quantitative real-time PCR (qRT-PCR) using OsActin as an internal reference gene. Data are presented as mean ± SE (n = 3 replicates per measurement). An asterisk (∗) indicates a statistically significant difference in relative gene expression (two-sided t-test; ∗P < 0.05, ∗∗P < 0.01).

We used bioinformatics to determine if Xosr001 in OMVs could form stable secondary structures and if it could interact with rice mRNAs (Supplemental Table 4). The complementary sequences of Xosr001 were aligned with the Nipponbare reference mRNA sequences at NCBI using BLASTN 2.2.31 (Sjöström et al., 2015) to identify complementary rice mRNAs. Matches with an E-value <12, combined with the RNA-seq results, were considered to be potential host immune targets of Xosr001. We were particularly interested in OsJMT1, which encodes a JA methyl transferase that is a crucial component of the plant immune response to pathogens and is responsible for converting JA to MeJA (Kim et al., 2009). Our bioinformatic analysis predicted that Xosr001 could target the coding sequence of OsJMT1 mRNA (Supplemental Figure 4A), and our RNA-seq results revealed that OsJMT1 expression was higher in leaves inoculated with ΔXosr001 compared to leaves inoculated with the WT strain (Figure 3A). To analyze the temporal changes in OsJMT1 expression, leaves of 6-week-old Nipponbare plants were inoculated with the WT strain, and tissues were collected at 10, 18, 24, 36, and 48 hpi. At 10 hpi, OsJMT1 expression was significantly reduced in leaves inoculated with the WT strain compared to non-inoculated leaves (Figure 3B). Interestingly, a substantial decrease in Xosr001 transcript abundance was observed at 36 hpi, whereas OsJMT1 was clearly higher at this timepoint compared to 10 hpi (Figure 3B). These results suggest that Xosr001 may promote the degradation of OsJMT1 transcripts. RT-qPCR and northern blot analyses revealed that OsJMT1 transcripts were more abundant after 10 hpi (Figure 3C). Collectively, these results suggest that the perception of OMV-packaged Xosr001 triggers a dysregulated response in the host plant.

Xosr001 impacts OsJMT1 expression and endogenous MeJA content

To evaluate the impact of Xosr001 on OsJMT1 transcript abundance in vivo and its occurrence within plant tissues, OMVs were isolated from ΔXosr001 (Supplemental Figure 4B). Leaves of 6-week-old Nipponbare rice plants were inoculated with purified OMVs from WT or ΔXosr001 strains, with WT-inoculated leaves serving as a negative control. As expected, leaves inoculated with ΔXosr001 had enhanced OsJMT1 transcription compared to leaves inoculated with the WT strain at 24 hpi (Figure 4A and A′). RNA-FISH was performed using RNA probes specific to Xosr001 and OsJMT1 to determine if OsJMT1 expression was suppressed by Xosr001 in vivo. The fluorescence intensity derived from OsJMT1 transcripts in WT-inoculated leaves was approximately 36% of the intensity in leaves inoculated with ΔXosr001 (Figure 4B). This result suggests that OMVs lacking Xosr001 were unable to suppress the expression of OsJMT1 in rice leaves. Additionally, OsJMT1 transcripts were observed in epidermal and mechanical tissues of rice leaves where they colocalized with OMV-packaged Xosr001 (Figure 4A). This indicated that Xosr001 suppressed OsJMT1 transcript accumulation only when it was transferred into epidermal and mechanical tissue of rice leaves.

Figure 4.

Figure 4

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OsJMT1 is the potential target of Xosr001

Localization of the OsJMT1 transcript (magenta) and Xosr001 transcript (green) using hybridization chain reaction fluorescence in situ hybridization labeling.

(A) Leaves of 6-week-old Nipponbare rice plants were inoculated with purified OMVs from WT and ΔXosr001 at 24 hpi.

(B) Quantification of the OsJMT1 signal using the relative fluorescence intensity of a Z-series image of the light organ (n = 5). P values were calculated using one-way ANOVA with TMC (∗∗P < 0.01).

(C) Endogenous MeJA level of rice leaves. Leaves of 6-week-old Nipponbare rice plants were inoculated with WT, ΔXosr001, and ΔXosr001-pXosr for 24 hpi.

(D) Relative transcriptional levels of OsJMT1 after inoculation with H2O, sense Xosr001, and anti-sense Xosr001 for 0, 10, or 18 hpi. Gene expression was detected using quantitative real-time PCR (qRT-PCR) using OsActin as an internal reference gene. Data are presented as mean ± SE (n = 3 replicates per measurement).

(E) Northern blot and western blot analyses of OsJMT1 expression were performed by inoculating leaves with Xoc BLS256 for 0, 10, or 18 hpi. 5S rRNA and OsActin were used as loading controls. The values above each band represent band intensity and were calculated using ImageJ software. Band intensity in the first lane is normalized to 100. All data are presented as mean ± SEM. An asterisk (∗) indicates significant differences at P < 0.05 (ANOVA and Duncan’s multiple range test).

To further characterize Xosr001-mediated regulation of OsJMT1, Xosr001 RNA was synthesized in the sense and anti-sense direction and exogenously applied to the leaves of OsJMT1-HA-OE transgenic lines using mechanical inoculation for 10 and 18 h, which expressed OsJMT1-HA driven by a CaMV 35S promoter. RT-qPCR and northern blot analyses revealed a significant reduction in OsJMT1 transcript abundance after the application of the sense strand of Xosr001 RNA compared to the anti-sense RNA and H2O negative controls (Figure 4D and 4E). Furthermore, OsJMT1-HA expression was highly reduced when exogenous Xosr001 was inoculated on rice leaves (Figure 4E).

Previous studies have demonstrated that OsJMT1 converts JA to MeJA (Kim et al., 2009). To determine if Xosr001 impacts the accumulation of endogenous MeJA, we complemented the ΔXosr001 mutant strain with a functional copy of Xosr001 expressed from a plasmid with a lac promoter (hereafter called ΔXosr001-pXosr). Compared to leaves inoculated with the WT strain, leaves inoculated with ΔXosr001 accumulated significantly more MeJA at 24 hpi, whereas no significant difference was observed in leaves inoculated with ΔXosr001-pXosr (Figure 4C). Our findings suggest that Xosr001 suppresses OsJMT1 transcript accumulation, which leads to a reduction in MeJA accumulation.

Xosr001 suppresses stomatal immunity to enhance disease susceptibility in rice

MeJA can induce stomatal immunity in plants to prevent phytopathogenic bacteria from entering host tissues during the initial infection (Melotto et al., 2017). We quantified the stomatal conductance (Gs) of rice leaves after a spray inoculation to determine if Xosr001 compromises stomatal immunity. Rice seedlings treated with water had significantly higher Gs values than those treated with any of the Xoc BLS256 strains or E. coli at 1 day post-inoculation (dpi) (Figure 5A). At 2 dpi, a substantial decrease in Gs occurred in leaves inoculated with ΔXosr001 compared to leaves inoculated with WT or ΔXosr001-pXosr. We found that E. coli-inoculated rice leaves had no significant difference in Gs after 1 and 2 dpi (Figure 5A). These findings suggest that Xoc BLS256 can promote stomatal opening, whereas the ΔXosr001 mutant is clearly impaired in its ability to induce stomatal opening. Furthermore, nonpathogenic E. coli are also unable to induce stomatal immunity in a nonhost plant.

Figure 5.

Figure 5

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Xosr001 enhances disease susceptibility by suppressing stomatal immunity in rice leaves

(A) Stomatal conductance in the leaves of 6-week-old seedlings from Nipponbare rice plants was performed after spray inoculation with WT, ΔXosr001, ΔXosr001-pXosr, E. coli, and water. Stomatal conductance was measured at 1-day post-inoculation (dpi) and 2 dpi. Data are shown as mean ± SE (n = 10 technical replicates per measurement). Lesion lengths (B) and disease lesions (C) on the WT-, ΔXosr001-, and ΔXosr001-pXosr-inoculated rice leaves after spray inoculation. Six-week-old seedlings were sprayed with 0.01% Silwet L77. Photographs were taken at 4 dpi. All data are presented as mean ± SD. ANOVA was performed with Dunnett’s multiple comparison post-hoc correction compared to the WT.

(D) Bacterial population sizes in the WT-, ΔXosr001-, and Δxosr001-pXosr-inoculated leaves. Bacterial populations were determined at 4 dpi.

(E) OsJMT1-HA was obviously degraded during Xoc BLS256 infection but remained relatively stable after ΔXosr001 infection. Three-week-old transgenic seedlings constitutively expressing OsJMT1-HA were sprayed with the strains Xoc BLS256, ΔXosr001, and ΔXosr001-pXosr. OsJMT1-HA was detected by immunoblotting at the indicated time points post inoculation.

(F) Virulence was assessed by inoculating 3-week-old susceptible rice plants. Leaves (n = 10) were inoculated with needleless syringes, and lesion lengths were evaluated 14 days after inoculation. All data are presented as mean ± SD. ANOVA was performed with Dunnett’s multiple comparison post-hoc correction compared to the WT (∗P < 0.05).

(G) Bacterial titers in the Xoc BLS256- and ΔXosr001-inoculated leaves of the OsJMT1-HA-OE and OsJMT1-KO transgenic plants. Bacterial titers were determined at 4 dpi. All data are presented as mean ± SE (n = 3 technical replicates per measurement). (D and G) Statistically significant differences in bacterial titers were determined using one-way ANOVA and Tukey’s honest significance test.

(H) Stomatal conductance in the leaves of the 3-week-old OsJMT1-HA-OE transgenic lines (OsJMT1-HA-OE-3 and OsJMT1-HA-OE-7) after challenge with Xoc BLS256 and ΔXosr001. Stomatal conductance was measured at 2 dpi. All data are presented as mean ± SE (n = 10 technical replicates per measurement).

Collectively, Xosr001 suppresses Xoc-mediated stomatal opening on rice leaves. To determine if Xosr001 contributes to disease susceptibility in rice, the leaves of 6-week-old Nipponbare plants were spray-inoculated with WT, ΔXosr001, or ΔXosr001-pXosr strains. The virulence of ΔXosr001 was clearly impaired as this strain was unable to produce lesions of the same length as those produced by the WT and ΔXosr001-pXosr strains (Figure 5B and 5C). Consistent with the lack of disease symptoms, the in planta titer of WT strainwas significantly greater than that of ΔXosr001 (Figure 5D). These results indicate that Xosr001 contributes to virulence in rice leaves by suppressing stomatal immunity.

We generated OsJMT1-HA-OE transgenic rice lines and used CRISPR/Cas9-based gene editing to create an OsJMT1 knockout mutant (OsJMT1-KO). These lines were used to provide additional evidence for the observation that Xosr001 delivered into host plant cells by OMVs impedes OsJMT1 transcript accumulation to suppress stomatal immunity. OsJMT1-HA expression in the transgenic lines was detected by western blot (Supplemental Figure 5). Spray inoculation with the WT and ΔXosr001-pXosr strains resulted in the degradation of OsJMT1-HA transcripts in OsJMT1-HA-OE transgenic seedlings, whereas OsJMT1-HA transcript accumulation was unaffected by the ΔXosr001 strain (Figure 5E). OsJMT1-HA-OE transgenic plants inoculated with ΔXosr001 exhibited significantly shorter lesions and lower bacterial titers than those inoculated with the WT strain, whereas the ΔXosr001 and WT strains exhibited no obvious differences in virulence when inoculated on OsJMT1-KO plants (Figure 5F and 5G). In the absence of Xosr001, Xoc BLS256 was significantly compromised in its ability to induce stomatal closure in OsJMT1-HA-OE transgenic plants (Figure 5H). Our findings suggest that Xosr001 reduces the accumulation of OsJMT1 transcripts to induce stomatal opening during infection.

Discussion

In this study, we demonstrate for the first time cross-species communication by sRNAs contained in phytopathogenic bacterial OMVs. Our study demonstrates that Xosr001 packaged in OMVs by Xoc BLS256 is a novel way of using nucleic acids to modulate host plant immune responses.

Recently, several studies have described microbial-derived RNA that can act as a PAMP to facilitate infection by hijacking the RNA interference machinery of the host (Koeppen et al., 2016; Choi et al., 2017; Tan et al., 2018). In light of these reports, vesicle-mediated delivery packages sRNAs into host tissues where they can regulate gene expression by pairing with specific regions of specific host transcripts. The periodontal pathogen Porphyromonas gingivalis drives sRNA23392. This 20-nt sRNA is delivered by OMVs and targets desmocollin-2 to promote the invasion and migration of oral squamous cell carcinoma cells (Liu et al., 2021). With the development of bioinformatic approaches, long sRNAs of 30-100 nt have been identified as enriched in bacterial OMVs using high-throughput sequencing (Ghosal et al., 2015; Moriano-Gutierrez et al., 2020). In this study, we identified sRNA Xosr001 (77 nt) as enriched in Xoc BLS256 OMVs and capable of being delivered into host plant cells (Figures 1 and 2). This suggests that differential packaging of sRNAs constitutes a significant portion of the OMV-associated RNA, although the mechanism by which sRNAs are packaged into OMVs remains largely unknown.

In plants, sRNAs from fungal plant pathogens, such as Botrytis cinerea, can be delivered into the host to regulate endogenous plant genes. These pathogens produce sRNAs (Bc-sRNAs) that enter the host cell and hijack the plant RNA interference machinery (Weiberg et al., 2013). This suggests that OMV-derived trans-kingdom gene silencing could occur between plants and phytopathogenic bacteria. In this study, we demonstrated that sRNA Xosr001 is highly abundant in OMVs of the phytopathogenic bacterium Xoc BLS256. RNA-FISH revealed that Xosr001 decreases the expression of OsJMT1 in rice leaves by blocking transcriptional processing (Figures 3 and 4). These results suggest that sRNA molecules can participate in communication between Xoc BLS256 and rice. While sRNAs associated with OMVs derived from Xoc BLS256 have not been reported previously, several groups have characterized the intracellular sRNA content of Xanthomonas using RNA-seq and have investigated the sRNA-mediated virulence regulatory pathway under different growth conditions (Liang et al., 2011; Tang et al., 2020; Wu et al., 2021). Tang et al. (2020) recently reported that a specific sRNA, RsmU, functions as a negative regulator of virulence and cell motility in Xcc. Moreover, our previous study found that sRNA Xonc3711 from Xoc plays an extensive role in oxidative stress and flagella formation by modulating the abundance of transcripts encoding the DNA-binding protein Xoc_3982 (Wu et al., 2021). Taken together, sRNAs could provide valuable insights into the virulence and biological circuitry of phytopathogenic Xanthomonas spp.

Disabling host stomatal immunity is required for some phytopathogenic bacteria to successfully colonize interior leaf tissues (Melotto et al., 2006). Several derivatives of JA are found naturally in plants (Staswick and Tiryaki, 2004). JA, and MeJA in particular, can induce the closure of stomatal pores (Suhita et al., 2004; Yan et al., 2015). Our results indicate that OMV-mediated delivery of Xosr001 is required by Xoc BLS256 to reduce the accumulation of OsJMT1 transcripts and to suppress endogenous MeJA accumulation during infection (Figure 4). We also demonstrate that the ability to induce stomatal closure is significantly compromised in ΔXosr001 compared to Xoc BLS256 after 2 dpi (Figure 5A). Notably, stomatal immunity and disease resistance were enhanced in the OsJMT1-HA-OE transgenic rice lines upon infection with ΔXosr001 compared to infection by Xoc BLS256 (Figure 5F–5H). These results indicate that Xosr001 inhibits the stomatal immunity of rice during infection. However, the mechanism by which Xosr001 directly targets OsJMT1 transcripts is still unknown. It is possible that sequence specificity or secondary structure play a role in the recognition of OsJMT1 by Xosr001.

OsJMT1-HA transcript degradation was observed in Xoc BLS256-inoculated rice leaves at 24 hpi (Figure 5E), suggesting Xosr001 facilitates OsJMT1-HA transcript degradation in stomata and other tissues. This finding is consistent with our observation that Xosr001 and OsJMT1 transcripts were detected in epidermal and mechanical tissues of rice leaves (Figure 4A and 4A′). In addition to inhibiting stomatal immunity, Xosr001 may have other functions in mechanical tissues. For instance, sRX061 is a homolog of Xosr001 that regulates multiple pathways, including virulence, the hypersensitive response, and swarming motility in Xcc (Tang et al., 2020). It would be very insightful for future studies to determine if Xosr001 also functions as a virulence effector through other mechanisms.

In summary, our research has uncovered a role for the OMV-mediated delivery of Xosr001 in interactions between pathogenic bacteria and host plants. Xosr001 can be packaged into OMVs of Xoc BLS256 and delivered into epidermal and mechanical tissues of rice leaves. This results in the inhibition of OsJMT1 transcription, suppression of endogenous MeJA accumulation, and suppression of stomatal immunity in rice (Figure 6). We present evidence that transmitting an sRNA signal via OMVs can modulate host defenses and is a key component of pathogen invasion and persistence. Further exploration is required to determine how Xosr001 directly interacts with OsJMT1 mRNA. We anticipate that host recognition of sRNAs will emerge as a major new category of communication between pathogens and the tissues they colonize.

Figure 6.

Figure 6

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Working model for Xosr001 function in inhibiting MeJA accumulation and suppressing stomatal immunity

During Xoc infection, the Xosr001 sRNA is secreted into host cells via OMVs. Upon entry into the host cell, Xosr001 inhibits the transcription of OsJMT1, which suppresses endogenous MeJA accumulation and causes stomata to remain open. This attenuates stomatal defense and allows the pathogen to successfully enter the host.

Methods

Strains, plasmids, and primers

The bacterial strains and plasmids used in this study are described in Supplemental Table 1. Primers used for the construction of mutant strains, plasmids, and DNA templates are provided in Supplemental Table 2.

Plant materials and bacterial strains

Oryza sativa ssp. japonica cv. Nipponbare was used as the WT plant and for generating transgenic plants. E. coli DH5α was cultured in Luria-Bertani medium at 37°C. Xoc BLS256 and derivative strains were grown in nutrient broth (NB) or NB containing 1.5% (w/v) agar (NA) as described previously (Nie et al., 2020). YEB medium (5 g/l yeast extract powder, 10 g/l Bacto tryptone, 5 g/l NaCl, 5 g/l sucrose crystallized, 5 g/l MgSO4·7H2O) and PSB medium (10 g/l peptone, 10 g/l sucrose crystallized, 1 g/l L(+)-glutamic acid, pH 7.4) were used for the release of OMVs by Xoc BLS256. Antibiotics were added to the medium at the following final concentrations: ampicillin (100 μg/ml), rifampicin (25 μg/ml), kanamycin (25 μg/ml), and spectinomycin (50 μg/ml).

Preparation of OMVs

OMVs were isolated as described previously (Mordukhovich and Bahar, 2017). In brief, starter cultures of Xoc BLS256 were created by inoculating two sterile tubes each containing 3 ml of sterile YEB medium with three to five colonies from a plate. The tubes were placed in a 28°C shaker overnight. Xoc BLS256 starters were then incubated at 28°C in 500 ml of PSB medium until the culture reached an OD600 of 0.6 for the isolation of OMVs. The cells were pelleted using low-speed centrifugation (8000 g) at 4°C, and the culture supernatant was filtered through a 0.22-μm PVDF membrane filter (Millipore). OMVs were then collected from the filtered supernatant by centrifugation for 2 h in a Beckman Coulter Optima XPN-100 ultracentrifuge at 180,000 g and 4°C. The OMV pellet was washed with OMV buffer (20 mM HEPES, 500 mM NaCl, pH 7.4) and re-pelleted by centrifugation at 200,000 g for 2 h at 4°C.

For purification of OMVs, the OMV pellets were re-suspended in OMV buffer in 60% OptiPrep Density Gradient Medium (Sigma-Aldrich) and layered with 0.8 ml of 40% OptiPrep, 0.8 ml of 35% OptiPrep, 1.6 ml of 30% OptiPrep, and 0.8 ml of 20% OptiPrep. Samples were centrifuged for 16 h at 100,000 g and 4°C. Five-hundred microliter fractions were removed from the top of the gradient. The OMVs resided in fractions 2 and 3, which corresponded to 25% OptiPrep, as previously described (Mordukhovich and Bahar, 2017). The resulting pellets were re-suspended in distilled water and filter-sterilized through a 0.22-μm PVDF membrane filter before being stored at -80°C.

RNase protection assay

A total of 100 ng of intact OMVs were incubated with 10 pg/μl RNaseA (Thermo Fisher Scientific) for 1 h at 37°C. Control OMVs were then incubated for 20 min at room temperature with 0.5% Triton X-100 (Sigma-Aldrich). OMVs were washed three times with PBS buffer to remove RNase. Total RNA was isolated with QIAzol lysis reagent (QIAGEN).

Nanoparticle tracking analysis

Nanoparticles in the isolated OMV suspensions were analyzed using the ZetaView PMX120 (Metrix) instrument. The platform was cleaned with 10 ml of distilled water three times to remove all residue and to remove any potential air bubbles before analyzing samples. The cell with the 100 nm alignment suspension was filled using sterile syringes for standardization and then cleaned with 10 ml of distilled water three times. OMV suspensions were diluted in distilled water to a final volume of 600 μl and injected into the sample chamber with sterile syringes, yielding particle concentrations of 106 particles per milliliter in accordance with the manufacturer’s recommendations. The software used for capturing and analyzing the data conformed with the ASTM E2834-12 standard.

TEM imaging

Isolated OMV samples were dropped onto copper grids for 2 min and treated with 3% (w/v) phosphotungstic acid for 30 s. The samples were analyzed using the Talos F200 transmission electron microscope (Thermo Fisher Scientific) set to 120 kV.

For TEM imaging, the rice leaf samples were placed in 2.5% (v/v) glutaraldehyde solution for at least 6 h, post-fixed in osmium tetroxide, dehydrated in a graded series of ethanol, and embedded in LR White embedding medium. Samples were polymerized at 60°C overnight and sectioned using the Leica EM UC7 microtome. The tissue samples were cut at 60-90 nm. TEM images were acquired using the Tecnai G2 Spirit Biotwin microscope operated at 120 kV.

RNA-seq analysis of Xoc BLS256 sRNAs

Purified OMVs from Xoc BLS256 were lysed using QIAzol reagent. OMV RNA was isolated using the miRNeasy Kit (QIAGEN), which retains the sRNA fraction. The RNA concentration was then determined using a bioanalyzer (Agilent Technologies). Total RNA (1 μg) was used to prepare the cDNA libraries using the TruSeq Small RNA Library Preparation Kit (Illumina). Libraries were sequenced using 50 bp single-end reads on a genome analyzer (Illumina). Reads were trimmed and aligned to the BLS256 reference genome (NC_017267) using CLC Genomics Workbench (CLC-Bio/QIAGEN). Data are deposited in NCBI under BioProject number PRJNA915483.

Construction of deletion and complementary mutants

The ΔXosr001 mutant strain was generated as described by Baba et al. (2006) with minor modifications. Two fragments flanking the target gene were amplified from the chromosomal DNA of Xoc BLS256 using Pfu polymerase (TransGen Biotech) and the primers described in Supplemental Table 2. The PCR products were digested, subcloned into the suicide vector pKMS1, and introduced into bacteria by electroporation (Bio-Rad Laboratories) with kanamycin selection. A single transformant with kanamycin resistance was selected, cultured for 8 h in NB, and inoculated as 10-fold dilutions on NA plates supplemented with 15% sucrose to obtain sucrose-insensitive clones.

To obtain the Xosr001 complementary mutant (ΔXosr001-pXosr), the full-length corresponding gene was amplified, and the fragment was cloned into pHM1 with the lac promoter. The recombinant plasmid was transferred into ΔXosr001 by electroporation, and transformants were screened on NA plates supplemented with spectinomycin.

RNA-seq and analysis of rice leaves

Total RNA was extracted from plant tissues at the site of inoculation using the MiniBEST Plant RNA Extraction Kit (TaKaRa) according to the manufacturer’s instructions. RNA was collected for two biological replicates for each condition. The concentration of total RNA was measured using the NanoDrop 3300 spectrophotometer (Thermo Fisher Scientific). mRNA was enriched from 4 μg of total RNA using magnetic beads linked with oligo(dT). The mRNA was fragmented and used for first- and second-strand cDNA synthesis. The resulting double-stranded cDNA was purified using AMPure XP beads. Purified double-stranded cDNA was then used for end repair, 3′ end adenosine tailing, sequencing adaptor connection, fragment selection, and finally PCR amplification to generate a sequencing library. The cDNA clusters were sequenced on the Illumina HiSeq 4000 platform using paired-end reads and sequencing by synthesis. The RNA-seq data were submitted to the NCBI database (https://www.ncbi.nlm.nih.gov/sra/) with SRA accession number PRJNA915483.

In vitro synthesis of sense and anti-sense Xosr001

Sense and anti-sense Xosr001 with a T7 promoter were prepared using 5 μg of DNA isolated from Xoc BLS256 by performing PCR with primers F/R-T7sense and F/R-T7antisense (Supplemental Table 2). The MAXIscript T7 In Vitro Transcription Kit (Thermo Fisher Scientific) was used to synthesize RNA from DNA templates, and sense and anti-sense Xosr001 RNA transcripts were purified with the MEGAclear Transcription Clean-Up Kit (Thermo Fisher Scientific). The RNA samples were diluted in 500 μl of water to a final concentration of 20 ng/μl. As a control, TRIS/EDTA buffer was used at a concentration equal to the final concentration of the RNA samples. A typical RNA concentration after elution was 500 ng/μl with a final TRIS/EDTA buffer concentration of 400 μM Tris–HCL and 40 μM EDTA. The mechanical inoculation of leaves was carried out as previously described (Lau et al., 2014; Dubrovina and Kiselev, 2019).

Virulence assays

Virulence assays were conducted using spray and pressure inoculation (Wang et al., 2021; Wu et al., 2021). For pressure inoculation, the bacterial concentration of each Xoc strain was adjusted to an OD600 of 0.8 and inoculated using needleless syringes. Lesion lengths were measured at 14 dpi. Twelve or more leaves were inoculated and evaluated for each Xoc strain. For spray inoculation, Xoc strains were suspended in 10 mM MgCl2 with 0.01% Silwet L77 to an OD600 of 0.8 and sprayed onto 4-week-old Nipponbare seedlings. After 4 dpi, three pieces of 5-cm-long leaves were detached from the inoculated seedlings, surface-sterilized with 75% (v/v) ethanol (Sigma-Aldrich), and ground in sterile water. Three technical replicates were collected for each sample. The samples were spread on NA plates after performing a serial dilution. Colony-forming units were counted two days after culturing.

Stomatal conductance measurement

Stomatal conductance was measured using a LI-6400XT photosynthesis system (Li-Cor) (Wang et al., 2021). In brief, stomatal conductance was measured in a 2 × 3-cm leaf chamber with a red-blue LED light set to 400 μmol mol−1 CO2 and 200 μmol m−2 s−1 photosynthetic photon flux density.

Transgenic constructs and plant transformation

OsJMT1 was amplified and cloned into pCAMBIA1300-35S::HA after digestion with EcoRI and HindIII. To construct the native promoter-driven expression vector, the full-length OsJMT1 gene (∼2 kb) was amplified from rice genomic DNA. The construct was transformed into A. tumefaciens strain EHA105 using the freeze-thaw method (Li et al., 2015). Transgenic rice plants were generated using Agrobacterium-mediated transformation of rice calli (Nipponbare cultivar). The regenerated shoots were transferred to 1/2×MS medium for rooting and were then planted in the greenhouse.

Nipponbare rice was genetically modified using CRISPR/Cas9 technology as previously described (Zhou et al., 2014). In brief, target sequences were selected near OsJMT1 (Supplemental Table 1), and the sgRNA was designed using CRISPR MultiTargeter (http://www.multicrispr.net/index.html) and synthesized by Shanghai Bioegene. The sgRNA and Cas9 constructs were transformed into Nipponbare callus by Agrobacterium-mediated transformation (Biorun).

RNA-FISH

Samples from rice leaves were placed in 50% FAA fixative solution (Sigma-Aldrich) for at least 24 h at 4°C. Fixed samples were re-hydrated by twice going through an ethanol series of 100%, 85%, and 75% (v/v) for 5 min each and DEPC-treated water for 1 min at room temperature. Samples were then embedded in LR White embedding medium and polymerized at 62°C for 2 h before being sectioned with the Leica EM UC7 microtome. After a protease (Sigma-Aldrich) digestion for 20 min at 37°C, the samples were treated with 0.2% glycine (Sigma-Aldrich) for 2 min, followed by TEA (Sigma-Aldrich), HCl, and acetic anhydride (Sigma-Aldrich) treatments. After two washes in PBS buffer, the samples were de-hydrated and hybridized with probes overnight at 53.3°C. For the symbiont Xosr001 transcript and OsJMT1 detection, RNA-FISH probes (Supplemental Table 2) were designed and provided by Molecular Instruments (Sangon Biotech). Images were adjusted to optimize the visual resolution using Leica Application Suite X (v. 3.4.2.18368).

MeJA extraction and quantification

Endogenous MeJA was extracted from 4-week-old rice leaves. In brief, 0.1 g of rice leaf tissues was ground to powder in liquid nitrogen. The powder was incubated overnight with 1 ml of PBS buffer (NaCl 8 g/l, KCl 0.2 g/l, Na2HPO4 3.63 g/l, KH2PO4 0.24 g/l, pH 7.4) at 4°C. The MeJA contents were quantified using the plant MethylJasmonate (MeJA) ELISA Kit (Mlbio BioTech) according to the manufacturer’s instructions.

Western blot

Total proteins were extracted from 0.1 g of rice leaf tissue by grinding the tissue in a mortar with liquid nitrogen until a fine powder was created. The leaf powder was then homogenized in 700 μl of extraction buffer (125 mM Tris–HCl, pH 6.8, 5% [w/v] sodium dodecyl sulfate, 10% [v/v] glycerol, and 2% [v/v] 2-mercaptoethanol). Total proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a PVDF membrane (Merck Millipore). OsJMT1-HA protein was detected by immunoblotting with an HRP-conjugated anti-HA monoclonal antibody diluted 1:2000 (Roche). OsActin was used as a loading control and was detected using an anti-actin monoclonal antibody diluted 1:5000 (CWBIO). The chemiluminescent signal was visualized using a Western Lightning Plus-ECL (Edo Biotech) and imaged using the ChemiScope 3000 mini instrument (CLiNX).

Northern blot

Total RNA was purified from Xoc strain liquid cultures (OD600 = 1.0) using the EasyPure RNA Kit (Transgen Biotech). Total RNA was extracted from plant leaves using the MiniBEST Plant RNA Extraction Kit (TaKaRa). RNA (10-20 μg) was separated using 1% agarose gels containing 25 mM guanidinium thiocyanate, transferred to Hybond N+ nitrocellulose membranes (Merck Millipore), and cross-linked to membranes by UV radiation. Probes were 5′ labeled with digoxygenin. Membranes were prehybridized for 10 min at 42°C, and then incubated with labeled probes overnight. Membranes were then rinsed, dried, and visualized by phosphorimaging on the ChemiScope 3000 mini instrument as previously described (Wu et al., 2021).

Quantitative real-time PCR

Quantitative real-time PCR was conducted as previously described (Wu et al., 2021). Gene expression was normalized relative to rpoD or OsActin using the ΔΔCT method, where CT is the threshold cycle. Three independent biological replicates were included and analyzed using the Wilcoxon-Mann-Whitney test.

Data and code availability

The RNA-seq data were submitted to NCBI database (https://www.ncbi.nlm.nih.gov/sra/) with SRA accession number PRJNA558244.

Funding

This work was supported by the National Natural Science Foundation of China (32272479, 32200142), Open Project Program of State Key Laboratory of Rice Biology (20190109), Open Project Program of State Key Laboratory for Biology of Plant Diseases and Insect Pests (SKLOF202201), Zhejiang Province Ecological Environment Research and Promotion Project (2020HT0009), Shanghai Committee of Science and Technology (19390743300 and 21ZR1435500), and Chongqing Natural Science Foundation (CSTB2022NSCQ-MSX0524).

Author contributions

Y.W., G.C., and B.Z. designed the experiments. Y.W., S.W., and P.W. performed most of the experiments. S.W. analyzed RNA-seq data. Y.W., W.N., and I.A. analyzed all other data. S.W. analyzed RNA-seq data. Y.W. wrote the article.

Acknowledgments

No conflict of interest is declared.

Published: January 12, 2024

Footnotes

Published by the Plant Communications Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and CEMPS, CAS.

Supplemental information is available at Plant Communications Online.

Contributor Information

Gongyou Chen, Email: gyouchen@sjtu.edu.cn.

Bo Zhu, Email: bzhu1981@sjtu.edu.cn.

Supplemental information

Document S1. Supplemental Figures 1-5 and supplemental Tables 1-3
mmc1.pdf (633.9KB, pdf)
Supplemental Table 4. The complementary sequences of Xosr001 were aligned with the Nipponbare rice cultivar reference mRNA sequences at NCBI using the algorithm BLASTN 2.2.31
mmc2.xlsx (54.4KB, xlsx)
Document S2. Article plus supplemental information
mmc3.pdf (3MB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Supplemental Figures 1-5 and supplemental Tables 1-3
mmc1.pdf (633.9KB, pdf)
Supplemental Table 4. The complementary sequences of Xosr001 were aligned with the Nipponbare rice cultivar reference mRNA sequences at NCBI using the algorithm BLASTN 2.2.31
mmc2.xlsx (54.4KB, xlsx)
Document S2. Article plus supplemental information
mmc3.pdf (3MB, pdf)

Data Availability Statement

The RNA-seq data were submitted to NCBI database (https://www.ncbi.nlm.nih.gov/sra/) with SRA accession number PRJNA558244.

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